INVESTIGATION Genetic Variants That Confer Resistance to Malaria Are Associated with Red Blood Cell Traits in African-Americans: An Electronic Medical Record-based Genome-Wide Association Study Keyue Ding,* Mariza de Andrade,† Teri A. Manolio,‡ Dana C. Crawford,§ Laura J. Rasmussen-Torvik,** Marylyn D. Ritchie,†† Joshua C. Denny,‡‡ Daniel R. Masys,§§ Hayan Jouni,*** Jennifer A. Pachecho,††† Abel N. Kho,††† Dan M. Roden,‡‡ Rex Chisholm,‡‡‡ and Iftikhar J. Kullo*,1 *Division of Cardiovascular Diseases, ***Department of Internal Medicine, and †Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota 55905, ‡Office of Population Genomics, National Human Genome Research Institute (NHGRI), Bethesda, Maryland 20892, ‡‡Departments of Biomedical Informatics, Medicine and Pharmacology, § and Center for Human Genetics Research, Vanderbilt University, Nashville, Tennessee 37232, ††Department of Biochemistry §§ and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, Department of Biomedical Informatics and Medical Education, University of Washington, Seattle, Washington 98195, ‡‡‡Center for Genetic Medicine, **Department of Preventive Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611 and †††Departments of Medicine and Preventive Medicine, Northwestern University, Chicago, Illinois 60611 ABSTRACT To identify novel genetic loci influencing interindividual variation in red blood cell (RBC) traits KEYWORDS in African-Americans, we conducted a genome-wide association study (GWAS) in 2315 individuals, divided red blood cell into discovery (n = 1904) and replication (n = 411) cohorts. The traits included hemoglobin concentration (RBC) traits (HGB), hematocrit (HCT), RBC count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), genome-wide and mean corpuscular hemoglobin concentration (MCHC). Patients were participants in the electronic MEdical association study Records and GEnomics (eMERGE) network and underwent genotyping of ~1.2 million single-nucleotide poly- African-Americans morphisms on the Illumina Human1M-Duo array. Association analyses were performed adjusting for age, sex, natural selection site, and population stratification. Three loci previously associated with resistance to malaria—HBB (11p15.4), informatics HBA1/HBA2 (16p13.3), and G6PD (Xq28)—were associated (P # 1 · 1026) with RBC traits in the discovery electronic medical cohort. The loci replicated in the replication cohort (P # 0.02), and were significant at a genome-wide signif- record icance level (P , 5 · 1028) in the combined cohort. The proportions of variance in RBC traits explained by significant variants at these loci were as follows: rs7120391 (near HBB) 1.3% of MCHC, rs9924561 (near HBA1/ A2) 5.5% of MCV, 6.9% of MCH and 2.9% of MCHC, and rs1050828 (in G6PD) 2.4% of RBC count, 2.9% of MCV, and 1.4% of MCH, respectively. We were not able to replicate loci identified by a previous GWAS of RBC traits in a European ancestry cohort of similar sample size, suggesting that the genetic architecture of RBC traits differs by race. In conclusion, genetic variants that confer resistance to malaria are associated with RBC traits in African-Americans. Disorders involving red blood cells (RBCs) are common and associated iron deficiency anemia, sickle-cell disease, and glucose-6-phosphate with adverse health outcomes (de Simone et al. 2005; Letcher et al. dehydrogenase (G6PD) deficiency, affect millions of people around 1983; Sarnak et al. 2002; Sharp et al. 1996). Such disorders, including the world and are a major cause of morbidity and mortality. RBC traits, including hemoglobin concentration (HGB), hematocrit (HCT), Copyright © 2013 Ding et al. RBC count, mean corpuscular volume (MCV), mean corpuscular doi: 10.1534/g3.113.006452 hemoglobin (MCH), and mean corpuscular hemoglobin concen- Manuscript received January 18, 2013; accepted for publication April 17, 2013 tration (MCHC), are commonly measured as part of the complete This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License (http://creativecommons.org/licenses/ blood count. The RBC traits have a substantial genetic component, by/3.0/), which permits unrestricted use, distribution, and reproduction in any with heritabilities of 0.56, 0.52, and 0.52 reported for RBC count, medium, provided the original work is properly cited. MCV, and MCH, respectively (Lin et al. 2007). Supporting information is available online at http://www.g3journal.org/lookup/ Genome-wide association studies (GWAS) have revealed multiple suppl/doi:10.1534/g3.113.006452/-/DC1. fl IRB 10-002758 EMR Phenotype/GWAS Consortium – Trait comparison loci that in uence interindividual variation in RBC traits in individ- 1Corresponding author: Mayo Clinic, 200 First Street SW, Rochester, MN 55905. uals of European and Asian ancestry (Ding et al. 2012; Ganesh et al. E-mail: [email protected] 2009; Kamatani et al. 2010; Kullo et al. 2010; Soranzo et al. 2009). Volume 3 | July 2013 | 1061 Whether these and additional novel loci influence RBC traits in indi- normal cardiac conduction led by Vanderbilt University Medical viduals of recent African ancestry is unknown. A GWAS for RBC Center (Denny et al. 2010) and a case-control study of type II traits in patients of recent African ancestry (i.e., African Americans) diabetes led by Northwestern University (Kho et al. 2012), respec- has yet to be reported. Identifying common genetic variants influ- tively. An additional 411 patients of African-American ancestry, encing RBC traits may offer insights into iron metabolism and enrolled at these sites for a GWAS of resistant hypertension, served erythropoiesis in African Americans, and such variants may also as the replication cohort. modify disease severity in conditions such as sickle-cell disease and thalassemia. It is well known for example, that sickle-cell hetero- Genotyping and quality control zygotes have less severe incidences of Plasmodium falciparum Genotyping was performed on the Illumina Human 1M-Duo platform infections than those with normal adult hemoglobin. Other abnor- at the Broad Institute of Harvard and Massachusetts Institute of mal hemoglobins, G6PD deficiency and pyruvate kinase deficiency Technology and for additional samples used for the resistant hyper- also confer some degree of resistance against falciparum malaria tension GWAS, at the Center for Inherited Disease Research at Johns (reviewed by Allison 2009). Hopkins University. The platform includes ~1.2 million markers with Differences in RBC traits between African Americans and non- a median spacing between markers of 1.5 kb (mean = 2.4 kb). These Hispanic white subjects have been observed in previous studies markers provide 76% coverage of the genome of an African population (Beutler and West 2005; Perry et al. 1992). Compared with non- at r2 . 0.8 (www.illumina.com/Documents/products/datasheets/ Hispanic white individuals, African Americans have lower hemo- datasheet_infiniumhd.pdf). Genotype data were cleaned using the globin levels, lower hematocrit levels, lower MCV, lower serum quality control (QC) pipeline developed by the eMERGE Genomics transferrin saturation, and greater levels of serum ferritin (Beutler Working Group (Turner et al. 2011; Zuvich et al. 2011). The process and West 2005). Such differences have been attributed to socio- includes evaluation of sample and marker call rate, gender mismatch economic, nutritional (Jackson 1990) and genetic factors (Perry and anomalies, duplicate and HapMap concordance, batch effects, et al. 1992). To identify novel genetic loci influencing interindivid- Hardy-Weinberg equilibrium, sample relatedness, and population ual variation in RBC traits, we conducted a two-stage GWAS in stratification. A total of 907,954 single-nucleotide polymorphisms 2315 African-American patients participating in the electronic (SNPs) in the Illumina 1M-Duo array were available for analysis MEdical Records and GEnomics (eMERGE) network. The network after we applied the following QC criteria: SNP call rate .98%, (www.gwas.org) was established by the National Human Genome sample call rate .98%, minor allele frequency .0.01, Hardy-Weinberg Research Institute to develop and implement approaches for leverag- equilibrium P .0.001, and 99.99% concordance rate in duplicates. ing biorepositories associated with EMR systems for large-scale One sample each from any related pairs was removed. After QC, genomic research (Kho et al. 2011; Manolio 2009; McCarty et al. 2315 patients African-Americans with phenotype and genotype data 2011). We have previously reported results of a GWAS for RBC were available for association analyses. traitsin3012patientsofEuropeanancestryintheMayoeMERGE cohort (Kullo et al. 2011), and in 12,486 patients of European Statistical analysis ancestry from the entire eMERGE network (Ding et al. 2012). When multiple measurements of a RBC trait were available for an fi We investigated whether loci identi ed in these and other cohorts individual patient, we chose the median value and the corresponding of European and Asian ancestry were associated with RBC traits in age for the genetic analyses. We performed association analyses by African-Americans. using linear regression implemented in PLINK (Purcell et al. 2007), assuming additive genetic effects, with adjustment for age, sex, site, fi METHODS and for any population substructure (i.e.,the rst two principal com- ponents [PCs]). We adjusted for